In Paroxysmal Nocturnal Hemoglobinuria (PNH) a number of hematopoietic cells cannot place or maintain glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface. This could be a result of defective GPI anchor synthesis or attachment or an overactive phospholipase. Our laboratory has begun to characterize the GPI biosynthetic pathway in mammalian cells. Several biosynthetic intermediates, including the complete GPI core, have been identified using a combination of metabolic labeling and chemical/enzymatic tests. A three-step procedure has been developed to provide unequivocal definition of biosynthetic defects in the mutants. First, [3H]mannose was used to label intact cells to identify the mannosylated GPI intermediates. Second, UDP-N-acetyl-[3H]glucosamine was used to label a cell-free system to identify earlier defects. Third, a complementation test was used to classify the mutants into different complementation classes. Here, we propose to apply this technology to a panel of murine and human mutant cell lines. The detailed structures of key GPI intermediates will analyzed with a combination of chromatographic and spectroscopic procedures, taking advantage of the accumulation of relatively high concentrations of intermediates in some mutants as a result of biosynthetic block. Similar analysis will be applied to PNH neutrophils and to PNH T cell lines and EBV-transformed B cells to pin-point the biosynthetic defect. Our research will lead to a better understanding of the biology of the GPI anchor in health and disease.